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...ScFvs, based on community demand. Figure 1: Antibody against PSD-95 from the Trimmer Lab. Information...addgene.org if you would like to contribute to the catalog. The Catalog Will Start With Neuroscience Antibodies ...performance in various applications are included in the full webpage. Educational Resources As part of...resource, which includes recombinant antibodies, educational materials, protocols, and shared data, on our...sequence information for the antibodies in our catalog, so that you will know exactly what you are working...community about how the antibodies work in various applications, enabling you to better plan your experiments... improving research infrastructure, from our educational materials to our repository of open samples. ...

...you do just this, probably in less than a minute (1). Green Listed is a new software tool used to design...found through https://wermelinglab.com/.   References 1. Panda, Sudeepta Kumar, et al. "Green listed—a CRISPR...the user. This approach is useful for several applications as will be discussed below. The software could...contain multiple gRNAs targeting all genes in the indicated species and were suggested to be the best gRNAs...

...them you can do today! 10 ways to share your science 1)   If you don’t already make it a habit to talk – ... Foundation Round Table. In any field, good communication skills help when it comes to finding a job. ...an argument for taking the time to hone your communication skills. So, where can a graduate student or ...opportunities than ever before for scientists to communicate about their work and their interest in science...your area or at your institution; the news or communications office is a good place to start. It’s not unusual...about science writing as a career, the Science Communication Program at the University of California, Santa... your research stories. Consider practicing communicating your research by guest blogging here, just leave...

...level of application not present in the online class.   Four ways to improve your next manuscript 1. Be consistent...of any scientist’s career. We use writing to communicate our findings and share them with the world. If...simply writing down your data doesn’t guarantee a publication. There is an art to composing manuscripts and...field of microbiology with multiple high-profile publications. My classmates consisted of graduate students...paper will be generated. After you complete a publication quality figure, produce the figure, its legend...

...and genome modifications to reporter vectors (e.g. pBV-Luc) and gene silencing (e.g. pLKO.1 - TRC cloning... (e.g. 6xHis containing pDEST-HisMBP). After purification the protein can be released from the resin and...types of yeast expression vectors. Consider your applications and experimental design. You can create a new...more traditional approach of an autonomously replicating vector that carries either a wild-type version...a variety of other types of vectors for most applications used to study gene function. From making transgenic...

...could be kept frozen after collection. Figure 1: Paul included a voltmeter on top of the battery box...under harsh conditions. One example application from the publication is the removal of unwanted cDNA derived...the van der Oost lab. Phylogenetic comparison indicates that like GeoCas9 and ThermoCas9, IgnaviCas9 is...a thermostable Cas9 be useful? It can enable applications where reagents must be stored at elevated temperatures...

...sequencing the unique DNA barcodes. Figure 1: Schematic of barcoding with a pooled library. Created...them useful for lineage tracing, among other applications. Cells can be identified using fluorescence,...sgRNAs to guide Cas9 to targeted genes. In these applications, however, nuclease-dead Cas9 is fused to a transcriptional...: Preparing Pooled Libraries Pooled Library Amplifications Resources on Addgene.org Browse all pooled... to Plasmid Pooled Libraries CRISPR Library Amplification Protocol ...

...year and happy reading! Top 10 blog posts of 2015 1. CRISPR 101: Homology Directed Repair Addgenie Chari...overjoyed to provide you with these fantastic educational resources and will be sure to keep our eyes open...used to generate inversions, deletions, and translocations. It also provides researchers with many different...repository use Cas9 to cut DNA, Addgene Science Communications Intern Mary Gearing explains how Cas9 homolog...make it easier to make gene edits at new genomic locations. 6. CRISPR 101: Non-homologous End Joining So ...

...help you decide if a master’s might be for you. 1. Are you interested in master’s-level jobs? Ultimately...technical know-how, and helped develop my science communication skills. Ultimately, my degree prepared me to...technical or product support specialist,” “field application scientist,” or “scientific or technical writer...for PhD applicants since many will also accept applications from master’s candidates with experience.  Use...

...specific DNA sequence in only 1 min. Using these lentiviral plasmids, catalytically inactive Cas9 (dSpyCas9)...makes them more adaptable to a broad range of applications. Recently, the Spiess lab developed a set of... with specific genomic loci. He made use of catalytically inactive dCas9 by fusing it to an engineered...

... to just that particular assay.      Fig. 1: A native protein (left) is denatured, allowing the...Conformation changes usually fall into one of two categories: ones induced by the researcher and ones that...naturally, by binding to other proteins, changing locations, or even just as part of performing a specific...it a transmembrane protein, or is it know to translocate or fold in certain conditions? It can help to...

...evidence through education.  My only criticisms of what I saw are that we, as a society, need to: 1) use more...impartial. The best science happens when scientists have 1) the courage to make mistakes and 2) the integrity...also a science communicator, UBC Public Scholar, and a member of the Vancouver SciCATs. Follow her on ...researchers and supporters of science marched to advocate for scientist participation in government, evidence...promote help motivate you to spend a little time advocating for science and getting yourself out there for...April because devaluing science has alarming implications for our public policies—many of which impact...scientist. I’ve worked tirelessly to get the publications, grant funding, and teaching experience that...

...targeting MNase to antibody bound proteins (Figure 1). Importantly, since Protein A has weaker affinity...interactions for subsequent sheering of DNA by sonification. Though it helps capture weaker or more transient...change offers a significant improvement and simplification of the original technique, specifically in ...

...updated version of BRET called BRET2. Figure 1: TRUPATH measures heterotrimeric G protein dissociation...Kadowaki S, Haga T, Takaesu H, Mitaku S (2002) Identification of G protein-coupled receptor genes from the...biosensors Resources on Addgene.org Browse the catalog of fluorescent protein plasmids Find biosensors...

...can also rank the plasmids by abundance, scaled to 1, to create a “cumulative wealth distribution” curve...would be a good time to talk about the amplification and verification processes we use to ensure high-quality...your own experiments. Library amplification 101 Library amplification seems straightforward: E. coli ...transformed with the library DNA, they grow and replicate, and then their DNA is harvested, much like one...library has its own depositor transformation and amplification protocol, but the overall workflow is similar...ll need to remember when undertaking library amplification and sequencing. Maintain representation of the...electrocompetent Stbl4 cells when performing amplifications, and we use electroporation to ensure a good...

...net negative charge with 4 negatively charged and 1 positively charged amino acids. Myc tags are frequently...(stay tuned for our affinity purification tag blog!).   Applications of epitope tags   The...success rates across all applications. You can find FLAG antibodies on Addgene’s catalog including a mouse monoclonal...protein purification is not ideal with Myc, as low pH conditions are required during the purification, which...experienced. Finding an antibody that works for your application, specifically detects your protein, is species...and have been widely used across species and applications. In this blog, we will review the most common...that are bound by antibodies for the purpose of purification or detection of the tagged protein. These peptides...

...References 1. Huang, Ally, et al. "BioBits™ Explorer: A modular synthetic biology education kit." Science...plasmids and their potential educational uses. We believe that biology education is something that should ... expensive tiny amounts of reagents are, how complicated (and again, expensive) the different machines...desire to create a set of low-cost, easy-to-use educational biology activities – that any school could afford...proteins without the need to handle a living, replicating organism. Furthermore, cell free systems can ...in adapting your research ideas into hands-on educational activities using the BioBits platform, please...BioBits™ Bright: A fluorescent synthetic biology education kit." Science Advances 4.8 (2018): eaat5107. PubMed...

...the brain where DA quantitation is desired (Figure 1). The probe perfuses liquid, which causes the passive...at a certain frequency, and this frequency can indicate behavioral state, for example, if someone is awake...

...into three main effector functions.  Figure 1: Fc effector functions in an immune response to a ...determination, don’t generally affect antibody applications like ELISAs or Western blots, they are key in...

...motion videos is very time consuming; producing a 1-2 minute video takes an entire week of our free time...Research 5 Reasons to Use Reddit for Science Communication 10 Ways to Share Your Science Resources on ...

... Table 1: New ready-to-use viral vectors in the Addgene repository  Name Serotype Category Depositor...

...enables the high-resolution tracking of more than 1 million cancer cells under drug treatment.” Stegmeier... interference. In the first two months since publication, the lab got over 100 requests for the plasmids...

...materials to the research community. References 1. Coroadinha, A. S., et al. "Effect of osmotic pressure...AAV yields. Given the wide range of downstream applications scientists rely on our AAV preparations for,...

...difficult to modify in the past.   Figure 1: Identification and enrichment use of XMAS-TREE. a) Cells ...  After identification of ABE activity, XMAS-TREE can also be used to purify these modifications and work...ABE-targeted cells By Ellie Adams Single base modifications through ABE or CBE's have the potential ability...point mutations. They allow single-nucleotide modifications to edit DNA without requiring a double-stranded...mCherry and GFP expression to check for ABE’s. b) Quantification of base-editing efficiency using XMAS-TREE-...methods at various genomic loci in four different categories.     Brookhouser, N., et al. BMC Biol 18...MYC-dependent cancer cell lines. It can be applied for identification of essential MYC binding sites in a wide range...

...vesicles. Okada CY, Rechsteiner M. Cell. 1982 May;29(1):33-41. PubMed. Protein transduction: unrestricted...protein-only transduction method and its potential applications to CRISPR/Cas9 genome editing.   Protein-only...the HIV protein Tat. Although CPP fusions can translocate a cell membrane, the presence of the CPP may ...that components in the buffer used for protein purification must be helping the untagged protein enter cells...Diego D'Astolfo. CRISPR/Cas9: a potential application of iTOP Having tested iTOP using Cre-lox recombination...showed up to 70% resistance to diphtheria toxin, indicating a 70% frequency of biallelic mutations. Importantly...Importantly, hESCs retained their stemness, indicating that iTOP may be valuable for CRISPR/Cas9 stem cell...

...do researchers track fluorescently labeled cells? 1. Epifluorescence microscopy This technique uses a ...of the immune system and its dynamics. References 1. Peters, Nathan C., et al. "In vivo imaging reveals... required for a growing organism to make the complicated transition from a clump of cells to a fully differentiated...relatively little tissue absorption, weak tissue scattering and small amounts of tissue auto-fluorescence...

...occur, but good performers move around easily  Table 1: Taking it a Step Further - Big Company v. Small Company...scientific and technical training is that these educational paths prepare you for a vast selection of career...tech transfer, management consulting, science communication, policy and the diverse options in nonprofit...science is much like industry but the “product” is publications—the pressure to produce is the same You get ...and teach students Collaborations may be less “complicated” - for example, easy to initiate a collaboration...is different about non-academia? Scientific publications are a nice to have, not a need to have Must ...every 3-6 years—a short and exciting ride Complicated matrix structures are common Small integrated...

...Article contributed by Susanna Bachle Figure 1: Initial steps in the CRISPR-pooled screening using...infection, because this setup allows for experimental replicates for each of the gRNAs. The idea was to assign...cassette is removed, the effector is expressed. By truncating one of the recombination sites in the construct...intermediate (d), and sparse (e) labeling of the calcium indicator, GCaMP6f, in the Drosophila optic lobe. Image ... reporter (blue) and stained with PI (pink) to indicate cell death. Image from Diessl et al., 2020. ...https://dx.doi.org/10.15698%2Fmic2020.04.713 Truncated TALE-fluorescent protein fusions as a DNA staining...protein to the DNA. They created plasmids for purification of tTALE-eGFP and tTALE-mCherry. In comparison...

...extension. Now, they split EB1 into two halves (Fig. 1): one containing the microtubule binding domain and...cells, and has potential use for a wide range of applications in cellular biology. Van Haren J et al. Nature...versatile in vivo fluorescent reporter for rapid quantification of base-editing and Cas9 cleavage activity ...plasmid that allows for rapid fluorescence-based quantification of base editors that utilize dCas9 fused to... The reporter constitutively expresses eGFP to indicate reporter abundance, and mCherry fluorescence is...frame-shift mutation within the mCherry reporter. Quantification of editing efficiencies simply involves tracking...

... contain the full length plasmid.   Figure 1: The full lentiviral plasmid is expected to be >12... (AmpR) and origins of replication (ori and f1 ori) which allow it to replicate.   How can I prevent...the bacterial selection marker and origin of replication. During bacterial culture, only the plasmid with...the antibiotic resistance gene and origin will replicate, and the transgene will be lost. Over time, bacteria...the recombined plasmid is still visible. This indicates that the DNA prep contains a mixture of the full...

...variants. This resulted in the construction of redCALWY-1, a red-shifted FRET sensor for zinc using variants...Neurobiology has been working on genetically encoded indicators of calcium and other small molecules since the...lymphocytes, too, making them a versatile tool for application in both neuroscience and immunology. Don’t be...imaging for beginners Griesbeck says his calcium indicators can be used in mouse, Drosophila, C. elegans,...give it a shot. “Beginners sometimes think it’s complicated, but actually it is very good,” he said. “It ...his team is now applying it to improve upon an indicator they developed and published in 2010 for visualizing...

...heat-fixed smear with the crystal violet stain for 1 minute. Rinse the slide briefly with tap water. Flood...ll be talking about the ins and outs of this classification system, including its history, biology, and...repeatable phenomenon, Gram began using it as a classification system. Eventually, any bacteria that retained...permeable to different classes of antibiotics. The classification system is also useful in a laboratory setting...bacteria type. The gram positive and negative classification system is incredibly helpful in visualizing...stepping into a lab.   Anna Kamens is a science communication summer intern at Addgene and a rising third-...

... cell types and physiological states. References 1. Ingolia, Nicholas T., et al. “Genome-wide analysis... While ribosome profiling co-opts the physical location of the ribosome, TRICK (Translating RNA Imaging...development, the osk-TRICK reporter was double labeled, indicating translational repression. During later stages...ribosomes after initiation, to calculate a ribosome translocation rate of ~3.5 codons/second. They also explored...Resources the Addgene Blog Learn More about SunTag Catch up on Your GFP Background Choose the Brightest Fluorescent...

...mature HA-tagged proteins in vivo. Nature Communications, 10(1). PubMed PMID: 31270320. PubMed Central ...antibodies for extracellular and intracellular applications: CDR grafting to stable frameworks and structure-based...

...Transcriptional Programs with CRISPR RNA Scaffolds. Cell, 160(1), 339-350 (2015). PubMed. Find the Plasmids @Addgene... interference (CRISPRi) model consists of a catalytically dead Cas9 protein (dCas9) that lacks endonuclease...library of characterized aptamers and supporting publication: http://www.freebase.com/base/aptamer Cruz-...

...heterodimerizes with CIBN upon blue light exposure (see Fig 1*). When the group targeted the enhancer region of ...plasmids at Addgene Opperman J et al., Nature Communications, 2019. PubMed PMID: 31346176. Using LADL to...light-activated-dynamic-looping (LADL) system. The group fused dCas9 to a truncated CIB1 protein from Arabidopsis (CIBN), which they...

...process is explained more fully in the animated Fig. 1 below. Normalization is a fundamental and often understated...smaller fragments that are then barcoded for identification. This mixture of barcoded fragments is known...of Toronto. He is an author of 20+ scientific publications and patents.   ...

...post about multiplexing with Cpf1.   Figure 1: Multiplexing allows researchers to express multiple...be used in a wide range of bacteria. References 1. Maddalo, Danilo, et al. “In vivo engineering of oncogenic.... Using multiplexed gRNAs can target multiple locations in the genome to modify, whether it be editing... use is pX333 from the Ventura lab. pX333, a modification of pX330, contains humanized wtCas9 and two ...thus marking cells that have undergone genomic modification. In this system, Cas9 must be supplied on a ...with Cpf1 Additional Resources on Addgene.org Catch Up on Your CRISPR Background with Our Guide Pages...

...present in the data such as synapsin, hsyn synapsin-1, human synapsin, hSynap. Matching We capture multiple...that allows category-specific, narrow searches. We defined three top level categories — catalog, educational... in a given category, and serve as a shortcut to a search scoped to that category. Catalog ID suggestions...prefer to browse and discover items in our catalog or educational resources through our main dropdown menu... of Addgene’s customer’s needs. Categories We created a categorization of our information in order to ...depositors — each with subcategories such as plasmids and viral preps for catalog, blog posts and protocols...80,000+ reagents and 700+ educational resources — including blog posts, protocols, guides, and collections...

...red light rapidly deactivates the system. Version 1 of this far-red/red light-responsive system is spread...consistent, and controllable pulses of light. Complicated patterns of light intensity and wavelength can... also be used to engineer new pathways with sophisticated gene expression controls. Most cell types do...light; these two systems can be coexpressed for sophisticated control of gene expression. An E. coli light-sensitive...; only two plasmids are required per system. Applications of these systems The large dynamic range of ...suitable for various scientific or engineering applications.   References Refactoring and optimization ...Oct 11. PubMed. Find the plasmids from this publication at Addgene. Synthetic biology: engineering Escherichia...

...instead conveniently provide them on your smart phone. 1. Lab Archives by LabArchives The single, most important...Gibco organizes its app into four categories: tools, analytics, education and data manager. The tools are...are five FREE science lab apps that have been educational and great for lab life. These apps prevent you...lab notebook. Lab Archives is a multipurpose application that allows you to share your data with colleagues...other nice features. This app also has a great educational section. This section holds the Gibco Cell culture...directions including which buffers to use and that indicates if a sequential digest is needed. The enzyme finder... to this app; the user can search by sequence, category of enzyme, or name to discover all the information...

...to do that than in the Addgene blog?  Fig. 1: One of our favorite content collections is our Help...Our educational content  Have you ever wondered how we choose topics for the Addgene blog? Or why we...everything else we do! The goal of Addgene’s educational content Addgene truly believes that sharing speeds...antibody and content team, we identified what applications should be included and what basic information... The guide was then assigned to our science communication specialist, Rachel, to write. Identify expertise...through an editing process and final check before publication.   Do research! Nearly everything we write entails...Guide figures. This requires a fair amount of communication between our content team and our graphic designer...

...intern to complete and present on.    Figure 1: Addgene's summer interns presenting their capstone...compiled a list of three options from the Addgene catalog. These were a miR30 cassette, Syntaxin3, and osa-miR528...

...large sequences and even entire genes. Figure 1: Mechanism of CAST systems. A Cas effector (Cascade...featured 20 mutations in TnsB, the subunit that catalyzes DNA insertion, along with both evolved and engineered...

...and cost for AAV genome sequencing.  Figure 1: The viral genome sequencing workflow. Using this...that could be used as a template for downstream applications (Berns, 1972). Using qPCR and restriction enzyme...

... After all I was only going to be interviewed for 1 hour so having a broad enough insight was going to...there a move with either promotions or further education may allow you to transition into various realms...

...-viral applications are also possible through the use of nanoparticles. [6].    References 1. Zhang ...2020.  “CRISPR Gene Therapy: Applications, Limitations, and Implications for the Future.” Front. Oncol...with a dominant mutation in a gene that causes cataracts could be rescued by coinjection into zygotes of...the technology in different labs for different applications.” Li’s team noted that CRISPR/Cas9 has been ... explore its potential in mice with a dominant cataract disorder caused by a single copy of a gene known....  “CRISPR Genome Editing Technology and its Application in Genetic Diseases: A Review.” Current pharmaceutical...

...faster. Years of science, done in weeks Figure 1: Sending out 80 FedEx envelopes in a single day --...including more physiologically-relevant systems, to catch all the important interactions. It’s also important... we found in our map have an impact on virus replication. While we and our collaborators are starting ... Nsp3 and Nsp16, and includes an additional catalytically dead version of the viral protease, Nsp5. Our... 2xStrep-tag for easy detection and affinity purification. All but one are in a pLVX vector background...coronavirus, one FedEx package at a time Since the publication of our paper, our group has been inundated with...sending plasmids to all who request them -- in locations all over the world, as far flung as the EU, Russia...

...into account, the digest pattern depicted in Lane 1 would be expected for a XbaI/NotI digest. The program... one half of naturally occurring restriction modification systems that prokaryotes use to protect themselves... has genes encoding one or a few restriction modification systems containing methyltransferases that add... phage for instance). Aside from restriction modification systems, DNA methylation also plays an integral...integral role in regulating genome replication, repairing mismatched basepairs or small indels that occur...methylases) are independent from the restriction modification systems, yet can still affect whether certain...specifically associated with any restriction modification systems, their sequences may overlap with restriction...

...defunct (but fun) plug-in called Maps.  Figure 1: See a starfish? This is actually a map of Joanne ...FiercePharma), or sign up to be alerted about their new publications via Pubmed, so you can congratulate them when...